DNA coding protein kinase

ABSTRACT

The invention provides a DNA comprising a base sequence encoding an amino acid sequence for a novel protein kinase, a plasmid containing the DNA, a transformant transformed with the plasmid, a process for producing the DNA, and oligonucleotide which hybridizes with the base sequence encoding a novel protein kinase.

This application is a 371 of PCT/JP96/00660 filed Mar. 15, 1996.

TECHNICAL FIELD

This invention relates to a novel DNA containing the consensus sequenceof a protein kinase catalytic domain. More specifically, the inventionrelates to a novel DNA containing a catalytic domain consensus sequencecharacteristics of serine/threonine kinase. The invention also relatesto a plasmid containing said DNA, a transformant having said plasmid, aprocess for producing said DNA, and an oligonucleotide capable ofspecific hybridization with said DNA.

BACKGROUND ART

The studies on the mechanism of signal transduction into eukaryoticcells in response to external stimuli have seen a rapid progress inrecent years.

For instance, it has been unravelled that intracellular signaltransduction is accomplished by a complex kinase-cascaded signaltransduction system in response to stimulation by growth factors such asa nerve growth factor (NGF) and an epidermal growth factor (EGF) and itis known that a growth factor receptor which is a kind of tyrosinekinases and an MAP kinase family which is a kind of serine/threoninekinases are both involved in the transduction mechanism (Williams, L.T.et al., Annu. Rev. Biochem., 1993, 62, 453-481; Boleu, J. B. Oncogene,1993, 8, 2025-2031; Davis, R. J., J. Biol. Chem., 268, 1993,14553-14556). It is also known that the phosphorylation anddephosphorylation of proteins with enzymes belonging to acycline-dependent kinase (CDK) family which is a kind ofserine/threonine kinases are involved in the regulation of cell cyclesand studies on the details of the mechanism are in progress (Nurse, P.,Nature, 344, 503-508, 1990; Pines, J., Trends Biochem. Sci., 19,143-145, 1994).

In addition, an SNF1 gene is known as one of the genes believed to playan important role in the metabolism of saccharides in yeast cells.

Further in addition, it has recently been reported that an AMP activatedprotein kinase (AMPK) gene cloned from the rat liver shows a high levelof homology in amino acid sequence to the SNF1 gene (Carling, D. et al.,J. Biol. Chem., 1994, 269, 11442-11448). It is found that the AMPKinactivates enzymes such as acetyl CoA carboxylase which catalyzes thefirst step of fatty acid synthesis, 3-hydroxy-3-methylglutaryl CoAreductase which is a key enzyme to the biosynthesis of cholesterol andother isoprenoid compounds, and hormone-sensitive lipase in a way ofphosphorylation and it is considered to be one of the importantregulatory factors in lipid metabolisms including the metabolism oftriglycerides or cholesterol esters (Clarke, P. R. et al., EMBO J.,1990, 9, 2439-2446). Thus, it has been suggested that the SNF1 family isheavily involved in the metabolic regulation of carbon compounds such assaccharides or lipids in eukaryotic cells.

As described above, many reports have suggested that the phosphorylationand dephosphorylation of proteins have important roles in the adjustmentof various cell functions and it has also been reported thatsimilarities exist in the structures of catalytic domains of theparticipating protein kinases (Steven K. Hanks et al., Science, 1998,Vol. 241, pp. 42-52).

In recent years, the cloning of cDNAs of novel protein kinases bymethods utilizing the above-noted structural similarties of proteinkinases has been described in many reports (see, for example, Holtzman,D. et al., Proc. Natl. Acad. Sci. USA, 1987, 84, 8325-8329; Hanks, S.K., Proc. Natl. Acad. Sci. USA, 1987, 84 388-393; and Andrew F. Wilks,Proc. Natl. Acad. Sci. USA, 1989, vol. 86, pp. 1603-1607). With theadvances in the studies in the field of interest, the analysis of thefunctions of novel protein kinase genes is in progress and themechanisms of some important intracellular signal transduction are beingelucidated but it can hardly be said that all mechanisms have beencompletely unravelled.

DISCLOSURE OF INVENTION

As described above, protein kinases obviously have diverse roles to playwithin cells. Therefore, isolating and identifying a novel proteinkinase gene are expected to be useful for various purposes such asunravelling the mechanism of signal transduction within cells and thedevelopment of therapeutics of diseases due, for example, to abnormalactivities of the protein kinase encoded by the gene.

An object of the invention is to provide a DNA encoding a novel proteinkinase, a plasmid having said DNA and a transformant obtained bytransforming a host with said plasmid. Another object of the inventionis to provide a process for producing said DNA. Yet another object ofthe invention is to provide an antisense DNA capable of specifichybridization with the sequence of said DNA by synthesizing anoligonucleotide based on the sequence of said DNA.

The present inventors synthesized oligonucleotides based on the aminoacid sequence of a region conserved as a common feature to certain kindsof protein kinases and screened human fetal liver derived cDNAs by meansof a PCR method using the synthesized oligonucleotides as primers,thereby successfully isolating a cDNA encoding a novel protein kinase.This has led to the accomplishment of the present invention.

Thus, the present invention provides a novel DNA encoding a proteinkinase. Specifically, the present invention provides a DNA comprising abase sequence encoding the amino acid sequence set forth under SEQ. ID.NO. 7 in the Sequence Listing, or a base sequence which has aninsertion, deletion or substitution added to a portion of said basesequence encoding the amino acid sequence of SEQ. ID. NO. 7, or basesequence hybridizing with either of said base sequences.

According to an embodiment of the present invention, there is provided aDNA comprising the base sequence set forth under SEQ. ID. NO. 2 in theSequence Listing.

The present invention further provides a plasmid having said proteinkinase coding DNA.

The present invention further provides transformants obtained bytransforming prokaryotic or eukaryotic cells with a plasmid having saidprotein kinase coding DNA.

The present invention further provides a process for producing saidprotein kinase coding DNA, which comprises the steps of:

(1) synthesizing cDNAs from human-derived mRNAs with reversetranscriptase;

(2) performing PCR using as templates the cDNAs syhthesized in step (1)and using as primers oligonucleotides based on the amino acid sequenceof a region conserved as a common feature to certain kinds of proteinkinases; and

(3) isolating positive clones by screening a cDNA library containinglong-stranded cDNAs using as probes the clones obtained in step (2).

The present invention further provides an oligonucleotide capable ofspecific hybridization with a base sequence encoding the amino acidsequence set forth under SEQ. ID. NO.7 in the Sequence Listing.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 shows the result of Northern blot analysis of pLKB-1.

BEST MODE FOR CARRYING OUT THE INVENTION

The protein kinase gene of the invention encodes a sequence of 433 aminoacids and has a 1302-bp base sequence including a stop codon.

The DNA containing a gene encoding the amino acid sequence set forthunder SEQ. ID. NO. 7 in the Sequence Listing may typically be preparedby the following procedure.

First, poly(A)⁺ RNA is isolated from an organ of interest. In theexample to be described below, a human fetal liver was used; however, aslong as the gene of interest is expressed, the starting material is notlimited in any particular way. The isolation of RNA from the organ maybe performed by common procedures known to the skilled artisan, asexemplified by a guanidinium thiocyanate/hot phenol method, aguanidinium/cesium chloride method and a guanidinium hydrochloridemethod. The greater part of the thus obtained RNA fractions compriserRNA and tRNA which do not encode proteins and many of the mRNAs presentin the cytoplasm of eukaryotic cells are known to have a poly(A)sequence at the 3' end; hence, only poly(A)⁺ RNA is adsorbed on anoligo(dT)-cellulose column and then eluted to isolate the pure form ofpoly(A)⁺ RNA. Commercial grades of ploy(A)⁺ RNA derived from variousorgans are currently available (as from CLONTECH Inc.) and may besubstituted.

Subsequently, with the thus obtained poly(A)⁺ RNA used as a template,cDNAs are synthesized for use as templates in PCR. For the synthesis ofcDNA, common procedures known to the skilled artisan for preparing cDNAlibraries may be employed. Generally, a mixture comprising poly(A)⁺ RNA,reverse transcriptase, primers (e.g. oligo (dT) primer and randomprimer) and a buffer solution is incubated at an appropriate temperatureto synthesize a first cDNA strand; then, RNase and DNA polymerase areadded and the mixture is incubated at an appropriate temperature tosynthesize a second CDNA strand. In the usual case, the resultingdouble-stranded cDNA is cloned into an appropriate vector to prepare acDNA library; in the present invention, the thus obtained cDNAs may bedirectly used as templates to perform screening by PCR.

PCR (polymerase chain reaction) is a method in which a cycle consistingof three steps, denaturation of the template DNA, annealing of primersto a single strand of the template DNA and extension of a complementarystrand to the single-stranded template DNA starting from the annealedprimers, is repeated to amplify the DNA fragment in the region heldbetween the two primers.

As is apparent from this operating principle, the DNA sequence to beobtained (amplified) by PCR is largely dependent on the primers usedand, hence, determination of the primer sequence is an importantproblem. Since one of the objects of the present invention is to isolatea novel protein kinase gene, the present inventors noted the amino acidsequence of a region conserved as a common feature to certain kinds ofprotein kinases. They synthesized mixed primers T1 and T2 containing allcodons that correspond to said amino acid sequence and used them in theexample to be described hereinafter.

PCR may be performed by usual procedures. Specifically, a mixed solutioncontaining a template DNA, primers, a mixture of dNTPs, a buffersolution and Taq polymerase may be subjected to 20-40 repeated cycleseach consisting of incubations at a denaturation temperature (typically94°-96° C.) for 0.5-1 min, at an annealing temperature (typically37°-60° C.) for 0.5-2 min, and at an extension temperature (typically60°-72° C.) for 0.5-3 min. If necessary, different cycles may becombined.

The clones amplified by PCR are anticipated to contain the sequences ofthe employed primers at their termini. Hence, in order to check whetherthe clones obtained are novel, the PCR product may be subcloned into anappropriate vector and thereafter sequenced. In the present invention, aclone was obtained that had a novel DNA sequence as set forth under SEQ.ID. NO. 1 in the Sequence Listing given below. This clone had an overalllength of 186 bp and an open reading frame composed of 62 amino acidresidues could be taken out of this DNA sequence.

Further in the present invention, cloning of longer cDNAs was attemptedin order to investigate the structure of a more integral gene. Theprocedure of this cloning will be described below.

In order to clone longer cDNAs, short fragments of cDNA from the cDNAlibrary to be screened is preferably removed from the viewpoint ofscreening efficiency. Hence, in the present invention, theabove-described procedure was repeated to synthesize double-strandedcDNAs from human fetal liver poly(A)⁺ RNAs and thereafter subjected tosize fractionation by agarose gel electrophoresis so as to recover onlycDNAs of about 1 kbp or more, which were used to prepare the cDNAlibrary to be screened. Size fractionation may be performed prior to thesynthesis of double-stranded cDNAs, namely, at the stage of poly(A)⁺RNAs; in this alternative case, only RNA stretches beyond certainlengths can be recovered by suitable methods such as electrophoresis andprecipitation using sucrose density gradients.

From the thus fractionated double-stranded cDNAs, a cDNA library can beprepared by various procedures known to the skilled artisan. In theexample to be described below, a Lambda ZAP^(R) II vector system(Stratagene) was used from such viewpoints as the convenience ofprocedures in subsequent sequencing; needless to say, this is not thesole case of the invention.

In the next step, the clone obtained by the previous step of PCR whichcontained the novel DNA sequence is used as a probe to screen the cDNAlibrary, thereby attempting to isolate a clone encoding a more integralgene. Screening of the cDNA library may be performed by the ordinaryplaque hybridization method which is commonly known to the skilledartisan. Further, in order to remove false positive plaques which canpotentially be acquired in this plaque hybridization (primary screening)step, secondary screening may be performed either by repeating plaquehybridization or by applying PCR from such viewpoints as the ease ofoperations.

Subsequently, the insert (the portion containing the gene of interest)in each of the resulting positive clones is subcloned into a suitableplasmid vector to be preserved as a plasmid. The vector for cloning isnot limited in any particular way and may be selected as appropriate forthe purpose of subsequent operations. If desired, a suitable host may betransformed with the plasmid to prepare transformants for subsequentpreservation. The host to be transformed is not limited to anyparticular type as long as it is capable of conserving the plasmidstably and may appropriately be selected from among eukaryotic andprokaryotic cells depending upon the purpose of subsequent operations.E. coli is generally used as the host.

Since there exist in principle more than one codon which is composed ofthree base pairs to encode one amino acid, many DNA sequences exist forencoding a certain amino acid sequence. This is also true with the genethat has been shown by the present inventors to encode the amino acidsequence of a novel protein kinase and it should be noted that manypossibilities exist for DNA sequences other than that of the native genederived from human fetal liver which has been elucidated in the presentinvention. Thus, the DNA sequence of the present invention is in no waylimited to the native human derived DNA sequence but embraces other DNAsequences encoding the amino acid sequence of the novel protein kinaseelucidated in the present invention.

As is well known in the gene recombinant technology, a certain DNAsequence may be mutated without greatly changing its intrinsiccharacteristics (the protein kinase activity in the case of the presentinvention) of the protein encoded by the DNA sequence or in such a wayas to improve said characteristics. Therefore, any skilled artisan canoccasionally perform artificial insertion, deletion or substitution onthe DNA to be provided by the present invention without greatly changingits intrinsic characteristics or in such a way as to improve saidcharacteristics. The present invention also embraces such mutant genes.

The present invention further provides a DNA containing a base sequencethat hybridizes either with a base sequence encoding the amino acidsequence set forth under SEQ. ID. NO. 7 in the Sequence Listing or witha base sequence obtained by adding an insertion, deletion or asubstitution to a portion of that base sequence in the manner describedabove. Hybridization may be performed by applying commonly adoptedconditions for plaque hybridization (as described in Maniatis, T. etal., Molecular Cloning, Cold Spring Harbor Lab., 1989).

Further in addition, an antisense DNA can be prepared on the basis of abase sequence encoding the amino acid sequence set forth under SEQ. ID.NO. 7 in the Sequence Listing according to the invention. An antisenseDNA is a DNA that has a base sequence complementary to the mRNAgenerated by transcription from the DNA sequence of the invention. Whenincorporated into a cell, the antisence DNA may occasionally react withthe mRNA to form a hybrid, thereby retarding the synthesis of the finalproduct protein. The present invention also provides an oligonucleotidecapable of specific hybridization with a DNA sequence encoding the aminoacid sequence set forth under SEQ. ID. NO. 2 in the Sequence Listing.

The term "oligonucleotide" as used above should be understood in itsbroadest sense. Thus, it covers both (1) oligonucleotides containingnaturally occurring bases and sugar portions bound by phosphodiesterbonds, and (2) oligonucleotide analogues which function in similar waysto the oligonucleotides (1) but which have non-naturally occurringportions. Exemplary oligonulceotide analogues include thoseoligonycleotides which have been chemically modified in the phosphategroups, sugar portions, 3' and 5' ends with a view to providing improvedstability. If desired, phosphodiester bonds may be replaced by nonionicand nonchiral other structures. Also included within the category ofoligonucleotide analogues are those oligonucleotides which containmodified bases, namely, purine or pyrimidine other than those whichoccur naturally.

The length of oligonucleotides to be provided by the present inventionis not limited in any particular way but they have preferably 8-40, morepreferably 15-30, subunits (one subunit is a combination of a base and asugar and adjacent subunits are bound by phosphodiester bonds and thelike).

Preferred target sites of the mRNA with which the oligonucleotides to beprovided by the present invention will hybridize include a transferinitiation site, a translation initiation site, an intron/exon bindingsite and a 5' capping site. Considering the secondary structure of themRNA, it is more preferred to select sites with little or no sterichindrance.

The oligonucleotides of the present invention can be produced bysynthesis methods known to the skilled artisan, for example, by asolid-phase synthesis method using a DNA synthesizer typically availablefrom Applied Biosystems. Oligonucleotide analogues can also be producedby similar procedures (Akira Murakami et al., "Synthesis of FunctionalAntisense DNA" in Yuki Goseikagaku, 48(3): 180-193, 1990).

EXAMPLE

The following example is provided for the purpose of furtherillustrating the present invention but is in no way to be taken aslimiting.

(A) Searching for Novel Clones by PCR Using the cDNAs Derived from HumanFetal Liver Poly(A)⁺ RNA as templates:

A-1. Synthesis of PCR primers:

The inventors asked Sawady Technoloby Co., Lt. to synthesizeoligonucleotides T1 and T2 having the following sequences determined onthe basis of the amino acid sequence of a region conserved as a commonfeature to certain kinds of protein kinases. The prepared mixture of T1and T2 primers was used as a primer in PCR. The base sequence of T1primer is set forth under SEQ. ID. NO. 3 in the Sequence Listing andthat of T2 primer is set forth under SEQ. ID. NO. 4 in the SequenceListing. ##STR1## A-2. Preparation of templates for PCR:

Using a TimeSaver™ cDNA synthesis kit (Pharmacia), cDNAs weresynthesized from the poly(A)⁺ RNA of human fetal liver (CLONTECH) inaccordance with the attached manual; the synthesized cDNAs were used astemplates for PCR.

The specific procedures were as follows. Human fetal liver poly(A)⁺ RNA(5 μg) thermally denatured by warming at 65° C. for 10 min was added toa reaction solution for first-strand synthesis consisting of murinereverse transcriptase, random hexamer primer and a reaction buffersolution and the mixture was incubated at 37° C. for 1 hour tosynthesize the first cDNA strand. The reaction solution was subsequentlyadded to a reaction solution for second-strand synthesis consisting ofE. coli derived RNaseH, E. coli derived DNA polymerase I and a reactionbuffer solution and the mixture was incubated first at 12° C. for 30min, then at 22° C. for 1 hour to synthesize the second CDNA strand. Thereaction mixture was subjected to phenol/chloroform extraction to removeproteins, then subjected to ethanol precipitation to recover thesynthesized cDNAs, which were dissolved in 50 μL of TE buffer solution(Tris-HCl, 10 mM; EDTA, 1 mM; pH 8.0) and used as template DNA for PCR.

A-3. Searching for novel DNA by PCR:

The primers synthesized in A-1 and the template DNA prepared in A-2 wereincorporated in a reaction solution of the following recipe and PCR wasperformed with this solution.

Reaction Solution:

Template DNA: 2.5 μL (out of 50 μL of the DNA solution prepared in A-2)

T1 primer: 200 pmol

T2 primer: 200 pmol

dNTPs: each dNTP at a final concentration of 0.2 mM

Tap polumerase: 2.5 U

10×buffer solution: 10 μL

Sterilized water: to make 100 μL

After denaturation at 94° C. for 6 min, PCR was performed through 40cycles, each consisting of denaturation at 94° C. for 1 min, annealingat 48° C. for 1 min and extension at 72° C. for 2 min.

A-4. Subcloning and sequencing of PCR product:

After agarose gel electrophoresis of the PCR product obtained in A-3, aband of a predicted size (ca. 180 bp) was excised and DNA was recoveredby the glass beads method using Sephaglas™ BandPrep kit (Pharmacia).Using SureClone™ ligation kit (Pharmacia), the recovered DNA wassubcloned into a plasmid vector as follows in accordance with theattached manual.

First, a Klenow fragment, polynucleotide kinase and 10×reaction buffersolution (2 μL) were added to the recovered DNA (ca. 100 ng); themixture was diluted with sterilized water to a total volume of 20 μL andincubated at 37° C. for 30 min to make both terminals of the DNAblunt-ended and phosphorylate the 5' end.

To the thus conditioned DNA, pUC18 vector (50 ng) dephosphorylated aftercleavage with SmaI, T4DNA ligase, DTT and 2×reaction buffer solution (10μL) were added; the mixture was diluted with sterilized water to a totalvolume of 20 μL and incubated at 16° C. for 2 hours to effect ligation.Using a quarter of the reaction solution (5 μL), competent E. colistrain JM109 (available from Wako Pure Chemical Industries Co., Ltd.)was transformed in accordance with common procedures known to theskilled artisan (as described in Maniatis, T. et al., Molecular Cloning,Cold Spring Harbor Lab., 1989).

The transformed E. coli cells were sown on an LB plate containingampicillin (100 μg/mL), X-Gal(5-bromo-4-chloro-3-indoyl-β-D-galactoside: 40 μg/mL) and IPTG(isopropyl-β-D-thiogalactopyranoside; 0.1 mM) and incubated at 37° C.overnight. From the colonies growing on the LB plate, 44 white coloniespresumably harboring plasmid DNAs containing inserts were selected andthe plasmid DNAs were extracted and the inserts were sequenced in themanner to be described below.

The extraction of plasmid DNAs from the white colonies was performedusing QIAprep-spin kit (Funakoshi) in accordance with the common alkaliSDS method described in the attached manual. With the extracted templateplasmid DNAs, the base sequences of the inserts were determined asfollows by cycle sequencing with PRISM™ Terminator Mix (AppliedBiosystems).

To about 1 μg of the DNAs, a sequencing primer (3.3 pmol) and PRISM™Termination Mix (9.5 μL) were added and the mixture was diluted to atotal volume of 20 μL and subjected to 25 cycles of PCR, each cycleconsisting of reactions at 96° C.×30 sec, 50° C.×15 sec and 60° C.×4min. The excess primer and the fluorescent dye in the reaction solutionwere removed either by gel filtration on Micro-Spin™ S-200HR column(Pharmacia) or by several extractions with phenol/chloroform, and theDNAs were recovered by ethanol precipitation. The base sequences of therecovered DNAs were determined by electrophoresis and analysis with anApplied Biosystems model 373A Sequencer.

A-5. Analysis of clones:

Thirty of the 44 selected clones had an identical sequence to a knownprotein kinase with the reported structure; however, the other 14 cloneswere verified to have an identical but yet to be reported novelsequence. One of such clones was designated LKB-1, having the basesequence set forth under SEQ. ID. NO. 1 in the Sequence Listing.

The base sequence of LKB-1 had an open reading frame region consistingof 186 bases and it encoded 62 amino acids (SEQ ID NO:8).

In the next place, an attempt was made to isolate longer clones from theCDNA library constructed from human fetal liver poly(A)⁺ RNA.

(B) Isolation and Structural Analysis of Long Stranded Clones of LKB-1:

B-1. Preparation of cDNA library derived from human fetal liver poly(A)⁺RNA:

Double-stranded cDNAs were synthesized from human fetal liver poly(A)⁺RNA as in A-2 using TimeSaver™ cDNA synthesis kit, except that thepoly(A)⁺ RNA was used in an amount of 2 μg and that an oligo(dT)₁₂₋₁₈primer was used for first-strand syntheis. The synthesizeddouble-stranded cDNAs were fractionated for size by agaroseelectrophoresis and only the cDNAs having sizes of at least about 1 kbpwere recovered. An EcoRI/NotI adaptor was linked to the cDNA ends andthe unreacted adaptor in the reaction solution was removed with a spuncolumn and the remainder was incorporated into a preliminarily EcoRIcleaved and dephosphorylated Lambda ZAP^(R) II vector (Stratagene). E.coli strain XL1-Blue was used as a host. The cDNAs incorporated into thevector were packaged using GIGAPACK^(R) II PACKAGING EXTRACT(Stratagene) in accordance with the attached manual. Statedspecifically, a freeze/thaw extract, a sonic extract and theincorporated cDNAs were mixed and incubated at 22° C. for 2 hours toeffect packaging, thereby yielding a cDNA library.

B-2. Screening of the CDNA library:

Primary screening of the CDNA library was performed by plaquehybridization in accordance with the customary procedures described inthe literature (e.g. Maniatis, T. et al., Molecular Cloning, Cold SpringHarbor Lab., 1989).

Specifically, about 2×10⁵ phage particles were sown on an LB agar plateand the resulting plaques were transferred to Hybond-N filter(Amersham), alkali denatured and illuminated with UV light to immobilizethe DNA on the filter. The filter was then incubated in a hybridizationsolution (50% formamide, 4×SSC, 50 mM of HEPES, pH 7.0, 10×Denhardt'ssolution, 100 μg/mL of thermally denatured salmon sperm DNA) at 42° C.for 3 hours to effect prehybridization. Subsequently, hybridization wasperformed by incubation at 42° C. for at least 16 hours together with ³²P labelled and thermally denatured probes (inserts in LKB-1).

The probes were radiolabelled using Random Primer DNA labelling kit(Ver. 2 of Takara Shuzo Co., Ltd.) in accordance with the attachedmanual. Specifically, about 100 ng of the insert isolated in pure formby agarose gel electrophoresis was denatured thermally; thereafter, T1and T2 primers (each 20 pmol), a Klenow fragment and 50 μCi of α-³²P!dCTP (3000 Ci/mmol) were added to a solution containing the thermallydenatured LKB-1 insert DNA and the mixture was incubated at 37° C. for30 min to prepare labelled probes.

After the hybridization, the filter was washed with washing solution A(2×SSC, 0.1% SDS) twice at room temperature for 15 min, subsequentlywith washing solution B (0.5×SSC, 0.1% SDS) first at 50° C. for 20 min,then at 55° C. for 20 min, and finally with washing solution C (0.2×SSC,0.1% SDS) at 60° C. for 20 min. After the washing operations, the filterwas dried and subjected to autoradiography.

As a result of such screening of about 2×10⁵ pfu of phage clones, threepositive signals were obtained.

A single-plaque derived phage suspension was prepared from thesuspension of phage particles containing the positive clones obtained bythe primary screening and the thus prepared phage suspension was used toperform secondary screening by PCR under the conditions set forth below.For use in the PCR, the following two primers were prepared. Theirsequences were based on the sequence of the DNA in the LKB-1 insert. Thebase sequence of S2 primer is set forth under SEQ. ID. NO. 5 in theSequence Listing and that of A1 primer is set forth under SEQ. ID. NO. 6in the Sequence Listing.

S2: 5'-TGAAGAAGAAGAAGTTGCGAAGGA--3'(SEQ ID NO:5)

A1: 5'-CCACCAGCTGGATGACATTTTTGT--3'(SEQ ID NO:6)

PCR reaction solution:

Phage suspension: 2 μL

S2 primer: 50 pmol

A1 primer: 50 pmol

dNTPs: each dNTP at a final concentration of 0.2 mM

Taq polymerase: 1.25 U

10×buffer solution: 5 μL

Sterilized water to make: 50 μL

After denaturation at 94° C. for 6 min, PCR was performed through 40cycles, each consisting of denaturation at 94° C. for 1 min, annealingat 50° C. for 1 min and extension at 72° C. for 2 min.

The PCR product was subjected to agarose gel electrophoresis and twophage clones producing a band of the predicted size (ca. 100 bp) werefound positive.

B-3. Subcloning of positive phase clones into plasmid vector:

The positive phage clones were excised into a plasmid (pBluescript^(R)SK(-) vector) by an ExAssist™ TM/SOLR™ system using a helper phageExAssist and E. coli strain SOLR. In accordance with the manual attachedto PREDIGESTED LAMBDA ZAP^(R) II/EcoRI/CIAP cloning kit (Stratagene), E.coli strain Xl1-Blue was infected with the positive phage clones and thehelper phage and cultivated at 37° C. for 2.5 hours so that the plasmidexcised into the culture solution was incorporated into E. coli strainSOLR. The resulting clones were designated pLKB1-1 and pLKB1-2.

The inserts in the pLKB1-1 and pLKB1-2 were analyzed for base sequenceas in A-4, therby determining the base sequence of the novel proteinkinase of the invention (pLKB-1). The determined sequence is set forthunder SEQ. ID. NO. 2 in the Sequence Listing.

B-4. Structural analysis of pLKB-1:

The base sequence of pLKB-1 contained an open reading frame encoding 433amino acids. The pLKB-1 also had sequences that agreed with theconsensus sequences of an ATP binding domain and a protein kinasecatalytic domain as set forth below.

Homology with the consensus sequence of ATP binding domain:

ATP binding domain consensus sequence:

Leu, Ile, Val!-Gly-Xaa-Gly-Xaa- Phe, Tyr, Met!- Ser, Gly!-Xaa-Val (SEQID NO:9)

pLKB-1 sequence:

Leu-Gly-Glu-Gly-Ser-Tyr-Gly-Lys-Val corresponding to residues 55-63 ofSEQ ID NO:7)

Homology with the consensus sequence of protein kinase catalytic domain:

Protein kinase catalytic domain consensus sequence:

1!-Xaa- His, Tyr!-Xaa-Asp- 2!-Lys-Xaa-Xaa-Asn- 1!- 1!- 1! (SEQ ID NO:10)

pLKB-1 sequence:

Ile-Val-His-Lys-Asp-Ile-Lys-Pro-Gly-Asn-Leu-Leu-Leu corresponding toresidues 172-184 of SEQ ID NO:7 (where 1! represents Leu, Ile, Val, Met,Phe, Tyr, Cys!; 2! represents Leu, Ile, Val, Met, Phe, Tyr!; Xaarepresents any amino acid).

With respect to the base sequence set forth under SEQ. ID. NO. 2 in theSequence Listing, a homology search was conducted against theGenBank^(R) database by the Lipman-Pearson method (Lipman, D. J. et al.,Scinece 227, 1985, 1435-1441); the proteins which were found to have thehighest level of homology (on the order of 30%) were AMPK belonging tothe SNFl gene family, MPA kinase, etc.

As described above, pLKB-1 had sequences in agreement with various knownconsensus sequences and in other areas, it also contained a region inagreement with a sequence highly conserved in a known protein kinase; inview of these facts it may as well be said that the protein encoded bypLKB-1 is a novel protein kinase.

As set forth above, the consensus sequence of a protein kinase catalyticdomain has lysine (Lys) at position 7 and it is well known that this isa characteristic feature of serine/threonine kinase. It is also knownthat thyrosine kinase is characterized by having either one of arginine,serine, threonine, alanine and cysteine at the same position 7.Therefore, the protein encoded by pLKB-1 can safely be considered to beserine/threonine protein kinase.

E. coli harboring the thus obtained clone pLKB-1 containing the basesequence set forth under SEQ. ID. NO. 2 in the Sequence Listing wasinternationally deposited, under accession Number FERM BP-5035, with theNational Institute of Bioscience and Human-Technology, Agency ofIndustrial Science and Technology, 1-3, Higashi 1 chome, Tsukuba-shi,Ibaraki-ken 305, Japan on Mar. 7, 1995.

(C) Northern Blot Analysis of pLKB-1

Nylon membranes on which poly(A)⁺ RNAs as prepared from various humanorgans and cultured cell lines have been transferred and immobilized areavailable from CLONTECH Inc. and are extensively used when studying theexpression distribution of certain genes in humans. The inventortherefore purchased such membranes (Human Multiple Tissue NorthernBlot.Human, Human II, Human Fetal II and Human Cancer Cell Line) fromCLONETECH and performed Northern blot analysis in accordance with thestandard protocol described in the manual.

Specifically, the area of pLKB-1 which included the coding region wasexcised with restriction enzymes and purified by agarose gelelectrophoresis. The pure product was labelled with α-³² P!dCTP by thestandard random hexamer method using the Ready to Go DNA labelling beadspurchased from Pharmacia. After thermal denaturation, the labelled clonewas warmed in ExpressHyb Hybridization Solution (purchased fromCLONTECH) at 68° C. together with a selected membrane to thereby effecthybridization. After the hybridization, the clone was washed with 2×SSCand 0.1% SDS several times at room temperature, followed by two washingswith 0.1×SSC and 0.1% SDS at 50° C. for 20 min. The membrane was driedand then subjected to autoradiography. The results are shown in FIG. 1.

In almost all of the tissues investigated, weak expression of about 3kbp occurred, except that a stronger expression occurred in HL-60 cells,with a fairly strong expression being observed in adult testis, adultskeletal muscle, fetal liver and K562 cells.

INDUSTRIAL APPLICABILITY

The protein kinase coding DNA of the invention is a novel clone that hasnot been known to date.

The DNA of the invention is believed to encode protein knases, inparticular, serine/threonine protein kinase and it has been found tohave comparatively high levels of homology to AMPK and MAP kinase asdemonstrated by the homology search the results of which have beendescribed in the Example.

AMPK is known to be a kinase taking part in the regulation of themetabolism of lipids or sugars and the protein kinase encoded by thenovel DNA sequence of the invention also has the potential to work as afactor taking part in the same kind of metabolic regulation. On theother hand, MAP kinase is known to be a factor that plays an importantrole in the kinase cascade for intracellular signal transduction fromreceptors such as ones of cell growth factors and, hence, the proteinkinase encoded by the novel DNA sequence of the invention also has thepotential to take part in such kinase cascade.

Thus, the protein kinase encoded by the novel DNA of the invention isbelieved to take part in the regulation of certain cell functions.Therefore, therapeutic methods effective against diseases due toabnormal activities of the protein kinase of the invention canpotentially be offered by taking various approaches, such as (1)suppressing the in vivo expression of the gene of interest byadministering an antisense DNA or RNA or the like that are based on thebase sequence of that gene, (2) introducing the gene of interest intothe human body as it is incorporated into a suitable vector such as aretroviral vector, (3) developing drugs, which act on transferregulating factors which regulate in vivo expression of the gene ofinterest and/or a promoter and the like of the same gene, and (4)developing drugs which either suppress or promote the activities of theprotein kinase encoded by the gene of interest.

As FIG. 1 shows, a particularly strong expression occurred in severaltissues and it is well known that in such tissues, highlyundifferentiated cells capable of autoreproduction such as blood stemcells in the fetal liver occur to take on a "stem cell system". Beingleukemia cells, HL-60 and K562 cells are believed to maintain acomparatively undifferentiated state and it is also well known that theycan be induced to become differentiated in response to certain kinds ofstimulant. On the other hand, the leukocyte fractions of peripheralblood which are also blood cells but are believed to be in adifferentiated state allow for only a faint expression of the gene ofinterest. Thus, the gene of interest has a characteristic expressionpattern in that a very strong expression occurs in undifferentiatedcells or in tissues abundant with undifferentiated cells but that a veryweak expression occurs in differentiated cells. This suggests thepossibility that the kinase encoded by the gene of interestphosphorylates a certain substrate protein to control thedifferentiation of cells. Further, one could induce the differentationof undifferentiated cells by inhibiting the activity of the kinase whichis highly expressed in those cells. It is well known that certain kindsof cancer cells can be treated by differentiation inducing therapy usingsuitable agents such as retinoic acid. Therefore, the development of aninhibitor of the kinase might contribute to combatting cancers usingthat inhibitor.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 186 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTTGCGGTTAAGATGTTGAAGAAGAAGAAGTTGCGAAGGATCCCCAAC48                            ValAlaValLysMetLeuLysLysLysLysLeuArgArgIleProAsn                              151015                                                                        GGGGAGGCCAACGTGAAGAAGGAAATTCAACTACTGAGGAGGTTACGG96                            GlyGluAlaAsnValLysLysGluIleGlnLeuLeuArgArgLeuArg                              202530                                                                        CACAAAAATGTCATGCAGCTGGTGGATGTGTTATACAACGAAGAGAAG144                           HisLysAsnValMetGlnLeuValAspValLeuTyrAsnGluGluLys                              354045                                                                        CAGAAAATGTATATGGTGATGGAATACTGCTGCTACGGCGAG186                                 GlnLysMetTyrMetValMetGluTyrCysCysTyrGlyGlu                                    505560                                                                        (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1302 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       ATGGAGGTGGTGGACCCGCAGCAGCTGGGCATGTTCACGGAGGGCGAG48                            MetGluValValAspProGlnGlnLeuGlyMetPheThrGluGlyGlu                              51015                                                                         CTGATGTCGGTGGGTATGGACACGTTCATCCACCGCATCGACTCCACC96                            LeuMetSerValGlyMetAspThrPheIleHisArgIleAspSerThr                              202530                                                                        GAGGTCATCTACCAGCCGCGCCGCAAGCGGGCCAAGCTCATCGGCAAG144                           GluValIleTyrGlnProArgArgLysArgAlaLysLeuIleGlyLys                              354045                                                                        TACCTGATGGGGGACCTGCTGGGGGAAGGCTCTTACGGCAAGGTGAAG192                           TyrLeuMetGlyAspLeuLeuGlyGluGlySerTyrGlyLysValLys                              505560                                                                        GAGGTGCTGGACTCGGAGACGCTGTGCAGGAGGGCCGTCAAGATCCTC240                           GluValLeuAspSerGluThrLeuCysArgArgAlaValLysIleLeu                              65707580                                                                      AAGAAGAAGAAGTTGCGAAGGATCCCCAACGGGGAGGCCAACGTGAAG288                           LysLysLysLysLeuArgArgIleProAsnGlyGluAlaAsnValLys                              859095                                                                        AAGGAAATTCAACTACTGAGGAGGTTACGGCACAAAAATGTCATCCAG336                           LysGluIleGlnLeuLeuArgArgLeuArgHisLysAsnValIleGln                              100105110                                                                     CTGGTGGATGTGTTATACAACGAAGAGAAGCAGAAAATGTATATGGTG384                           LeuValAspValLeuTyrAsnGluGluLysGlnLysMetTyrMetVal                              115120125                                                                     ATGGAGTACTGCGTGTGTGGCATGCAGGAAATGCTGGACAGCGTGCCG432                           MetGluTyrCysValCysGlyMetGlnGluMetLeuAspSerValPro                              130135140                                                                     GAGAAGCGTTTCCCAGTGTGCCAGGCCCACGGGTACTTCTGTCAGCTG480                           GluLysArgPheProValCysGlnAlaHisGlyTyrPheCysGlnLeu                              145150155160                                                                  ATTGACGGCCTGGAGTACCTGCATAGCCAGGGCATTGTGCACAAGGAC528                           IleAspGlyLeuGluTyrLeuHisSerGlnGlyIleValHisLysAsp                              165170175                                                                     ATCAAGCCGGGGAACCTGCTGCTCACCACCGGTGGCACCCTCAAAATC576                           IleLysProGlyAsnLeuLeuLeuThrThrGlyGlyThrLeuLysIle                              180185190                                                                     TCCGACCTGGGCGTGGCCGAGGCACTGCACCCGTTCGCGGCGGACGAC624                           SerAspLeuGlyValAlaGluAlaLeuHisProPheAlaAlaAspAsp                              195200205                                                                     ACCTGCCGGACCAGCCAGGGCTCCCCGGCTTTCCAGCCGCCCGAGATT672                           ThrCysArgThrSerGlnGlySerProAlaPheGlnProProGluIle                              210215220                                                                     GCCAACGGCCTGGACACCTTCTCCGGCTTCAAGGTGGACATCTGGTCG720                           AlaAsnGlyLeuAspThrPheSerGlyPheLysValAspIleTrpSer                              225230235240                                                                  GCTGGGGTCACCCTCTACAACATCACCACGGGTCTGTACCCCTTCGAA768                           AlaGlyValThrLeuTyrAsnIleThrThrGlyLeuTyrProPheGlu                              245250255                                                                     GGGGACAACATCTACAAGTTGTTTGAGAACATCGGGAAGGGGAGCTAC816                           GlyAspAsnIleTyrLysLeuPheGluAsnIleGlyLysGlySerTyr                              260265270                                                                     GCCATCCCGGGCGACTGTGGCCCCCCGCTCTCTGACCTGCTGAAAGGG864                           AlaIleProGlyAspCysGlyProProLeuSerAspLeuLeuLysGly                              275280285                                                                     ATGCTTGAGTACGAACCGGCCAAGAGGTTCTCCATCCGGCAGATCCGG912                           MetLeuGluTyrGluProAlaLysArgPheSerIleArgGlnIleArg                              290295300                                                                     CAGCACAGCTGGTTCCGGAAGAAACATCCTCCGGCTGAAGCACCAGTG960                           GlnHisSerTrpPheArgLysLysHisProProAlaGluAlaProVal                              305310315320                                                                  CCCATCCCACCGAGCCCAGACACCAAGGACCGGTGGCGCAGCATGACT1008                          ProIleProProSerProAspThrLysAspArgTrpArgSerMetThr                              325330335                                                                     GTGGTGCCGTACTTGGAGGACCTGCACGGCGCGGACGAGGACGAGGAC1056                          ValValProTyrLeuGluAspLeuHisGlyAlaAspGluAspGluAsp                              340345350                                                                     CTCTTCGACATCGAGGATGACATCATCTACACTCAGGACTTCACGGTG1104                          LeuPheAspIleGluAspAspIleIleTyrThrGlnAspPheThrVal                              355360365                                                                     CCCGGACAGGTCCCAGAAGAGGAGGCCAGTCACAATGGACAGCGCCGG1152                          ProGlyGlnValProGluGluGluAlaSerHisAsnGlyGlnArgArg                              370375380                                                                     GGCCTCCCCAAGGCCGTGTGTATGAACGGCACAGAGGCGGCGCAGCTG1200                          GlyLeuProLysAlaValCysMetAsnGlyThrGluAlaAlaGlnLeu                              385390395400                                                                  AGCACCAAATCCAGGGCGGAGGGCCGGGCCCCCAACCCTGCCCGCAAG1248                          SerThrLysSerArgAlaGluGlyArgAlaProAsnProAlaArgLys                              405410415                                                                     GCCTGCTCCGCCAGCAGCAAGATCCGCCGGCTGTCGGCCTGCAAGCAG1296                          AlaCysSerAlaSerSerLysIleArgArgLeuSerAlaCysLysGln                              420425430                                                                     CAGTGA1302                                                                    Gln                                                                           (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GTNGCNGTNAARATGYTNAA20                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       TCNCCRTARCARCARTAYTC20                                                        (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       TGAAGAAGAAGAAGTTGCGAAGGA24                                                    (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 24 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CCACCAGCTGGATGACATTTTTGT24                                                    (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 433 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetGluValValAspProGlnGlnLeuGlyMetPheThrGluGlyGlu                              51015                                                                         LeuMetSerValGlyMetAspThrPheIleHisArgIleAspSerThr                              202530                                                                        GluValIleTyrGlnProArgArgLysArgAlaLysLeuIleGlyLys                              354045                                                                        TyrLeuMetGlyAspLeuLeuGlyGluGlySerTyrGlyLysValLys                              505560                                                                        GluValLeuAspSerGluThrLeuCysArgArgAlaValLysIleLeu                              65707580                                                                      LysLysLysLysLeuArgArgIleProAsnGlyGluAlaAsnValLys                              859095                                                                        LysGluIleGlnLeuLeuArgArgLeuArgHisLysAsnValIleGln                              100105110                                                                     LeuValAspValLeuTyrAsnGluGluLysGlnLysMetTyrMetVal                              115120125                                                                     MetGluTyrCysValCysGlyMetGlnGluMetLeuAspSerValPro                              130135140                                                                     GluLysArgPheProValCysGlnAlaHisGlyTyrPheCysGlnLeu                              145150155160                                                                  IleAspGlyLeuGluTyrLeuHisSerGlnGlyIleValHisLysAsp                              165170175                                                                     IleLysProGlyAsnLeuLeuLeuThrThrGlyGlyThrLeuLysIle                              180185190                                                                     SerAspLeuGlyValAlaGluAlaLeuHisProPheAlaAlaAspAsp                              195200205                                                                     ThrCysArgThrSerGlnGlySerProAlaPheGlnProProGluIle                              210215220                                                                     AlaAsnGlyLeuAspThrPheSerGlyPheLysValAspIleTrpSer                              225230235240                                                                  AlaGlyValThrLeuTyrAsnIleThrThrGlyLeuTyrProPheGlu                              245250255                                                                     GlyAspAsnIleTyrLysLeuPheGluAsnIleGlyLysGlySerTyr                              260265270                                                                     AlaIleProGlyAspCysGlyProProLeuSerAspLeuLeuLysGly                              275280285                                                                     MetLeuGluTyrGluProAlaLysArgPheSerIleArgGlnIleArg                              290295300                                                                     GlnHisSerTrpPheArgLysLysHisProProAlaGluAlaProVal                              305310315320                                                                  ProIleProProSerProAspThrLysAspArgTrpArgSerMetThr                              325330335                                                                     ValValProTyrLeuGluAspLeuHisGlyAlaAspGluAspGluAsp                              340345350                                                                     LeuPheAspIleGluAspAspIleIleTyrThrGlnAspPheThrVal                              355360365                                                                     ProGlyGlnValProGluGluGluAlaSerHisAsnGlyGlnArgArg                              370375380                                                                     GlyLeuProLysAlaValCysMetAsnGlyThrGluAlaAlaGlnLeu                              385390395400                                                                  SerThrLysSerArgAlaGluGlyArgAlaProAsnProAlaArgLys                              405410415                                                                     AlaCysSerAlaSerSerLysIleArgArgLeuSerAlaCysLysGln                              420425430                                                                     Gln                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 62 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ValAlaValLysMetLeuLysLysLysLysLeuArgArgIleProAsn                              151015                                                                        GlyGluAlaAsnValLysLysGluIleGlnLeuLeuArgArgLeuArg                              202530                                                                        HisLysAsnValMetGlnLeuValAspValLeuTyrAsnGluGluLys                              354045                                                                        GlnLysMetTyrMetValMetGluTyrCysCysTyrGlyGlu                                    505560                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Leu, Ile or Val                   Xaa in positions 3 and 5 are any amino acid                                   Xaa in position 6 is Phe, Tyr or Met                                          Xaa in position 7 is Ser or Gly                                               Xaa in position 8 is any amino acid                                           (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       XaaGlyXaaGlyXaaXaaXaaXaaVal                                                   (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: peptide                                                   (ix) FEATURE:                                                                 (D) OTHER INFORMATION: Xaa in position 1 is Leu, Ile, Val,                    Met, Phe, Tyr or Cys                                                          Xaa in position 2 is any amino acid                                           Xaa in position 3 is His or Tyr                                               Xaa in position 4 is any amino acid                                           Xaa in position 6 is Leu, Ile, Val, Met, Phe or Tyr                           Xaa in positions 8 and 9 are any amino acid                                   Xaa in positions 11-13 are Leu, Ile, Val, Met, Phe,                           Tyr or Cys                                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      XaaXaaXaaXaaAspXaaLysXaaXaaAsnXaaXaaXaa                                       510                                                                           __________________________________________________________________________

I claim:
 1. An isolated DNA encoding a protein having a protein kinaseactivity, comprising a base sequence encoding the amino acid sequence ofSEQ ID NO:7, or a base sequence encoding a protein kinase whichhybridizes with said base sequence encoding the amino acid sequence ofSEQ ID NO:7 in a solution of 0.2×SSC and 0.1% SDS at 60° C.
 2. Anisolated DNA according to claim 1, comprising the base sequence of SEQID NO:2.
 3. A plasmid having the DNA of claim
 1. 4. A transformantobtained by transforming a prokaryotic or eukaryotic cell with theplasmid of claim
 3. 5. An oligonucleotide which hybridizes with a basesequence encoding the amino acid sequence of SEQ ID NO:7 in a solutionof 0.2×SSC and 0.1% SDS at 60° C.
 6. An isolated DNA encoding a proteinhaving a protein kinase activity, comprising a base sequence encodingthe amino acid sequence of SEQ ID NO:7, or a base sequence encoding aprotein kinase which hybridizes with said base sequence encoding theamino acid sequence of SEQ ID NO:7 in a solution of 50% formamide,4×SSC, 50 mM HEPES, pH 7.0, 10×Denhardt's solution, 100 μg/ml thermallydenatured salmon sperm DNA at 42° C.
 7. An isolated DNA according toclaim 6, comprising the base sequence of SEQ ID NO:
 2. 8. A plasmidcomprising the isolated DNA of claim
 6. 9. A transformant obtained bytransforming a prokaryotic or eukaryotic cell with the plasmid of claim8.
 10. An oligonucleotide which hybridizes with a base sequence encodingthe amino acid sequence of SEQ ID NO: 7 in a solution of 50% formamide,4×SSC, 50 mM HEPES, pH 7.0, 10×Denhardt's solution, 100 μg/ml thermallydenatured salmon sperm DNA at 42° C.